CN113774114A - A kind of nucleic acid analysis method and application - Google Patents
A kind of nucleic acid analysis method and application Download PDFInfo
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- 108091070501 miRNA Proteins 0.000 claims abstract description 4
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
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Abstract
本发明涉及基因检测技术领域，公开了一种基于框架核酸DNA和CRISPR/Cas12a系统的电化学生物传感器制备方法及其应用，特点是包括界面上纳米材料AuNFs合成、构建四面体FNAs修饰的界面、CRISPR/Cas12a体系切割、电化学生物传感器信号输出四个步骤，构建基于CRISPR/Cas12a和框架核酸DNA的电化学生物传感平台，优点是实现了高稳定性、高灵敏度，且通过设计从四面体FNAs延伸出的DNA探针序列，可广泛应用于其他病毒、游离肿瘤DNA、miRNA和疾病相关的生物标记物的分析检测中。
The invention relates to the technical field of gene detection, and discloses a preparation method and application of an electrochemical biosensor based on framework nucleic acid DNA and CRISPR/Cas12a system. The four steps of CRISPR/Cas12a system cleavage and electrochemical biosensor signal output are to construct an electrochemical biosensing platform based on CRISPR/Cas12a and framework nucleic acid DNA. The DNA probe sequences extended by FNAs can be widely used in the analysis and detection of other viruses, cell-free tumor DNA, miRNA and disease-related biomarkers.
Description
Technical Field
The invention relates to the technical field of gene detection, in particular to a CRISPR/Cas electrochemical nucleic acid analysis technology based on DNA framework nucleic acid regulation.
Background
Nucleic acid analysis is of great significance for the prevention, diagnosis and prognostic monitoring of diseases. Traditional nucleic acid analysis methods include isothermal amplification, microarray, next generation sequencing, gold standard quantitative polymerase chain reaction, etc., but these methods all require pre-amplification, special equipment and trained professionals to obtain accurate and reliable results, and have the defects of time consumption, high detection cost, etc. Therefore, it is necessary to develop a rapid, low-cost, easy-to-use, highly sensitive nucleic acid detection method.
The CRISPR/Cas is used as a component of a bacterial immune system and has wide application in gene editing. Due to its unique non-specific single-stranded nucleic acid cleavage activity, novel nucleic acid detection biosensing technologies based on CRISPR/Cas systems have been developed for nucleic acid detection. However, most diagnostic platforms based on CRISPR/Cas systems rely on the trans-cleavage of non-specific single-stranded DNA (ssdna) reporter probes, whereas single-stranded DNA reporters are usually double-labeled and comprise a fluorescent molecule and a quenching molecule, which are respectively suspended at two ends of single-stranded DNA, and the signal reading method mainly relies on a fluorescence detection device, which may cause false positive results and increase the cost of signal reading.
The electrochemical biosensing platform has the advantages of high sensitivity, short detection time, low price, easy integration and the like in the aspect of molecular diagnosis. However, the sensitivity of CRISPR/Cas-based electrochemical nucleic acid analysis techniques is still limited by the accessibility of the Cas protein and the target molecule to the corresponding probes immobilized on a heterogeneous electrode interface.
Disclosure of Invention
In view of the above-mentioned drawbacks of the prior art, it is an object of the present invention to provide a method for analyzing nucleic acid with high sensitivity, high selectivity, simple and rapid operation, and high integration.
The technical scheme provided by the invention is as follows: the method comprises the following steps of performing cis-trans cutting on a double-phase interface supported by FNAs (DNA framework nucleic acid) by using a CRISPR/Cas12a system to construct a novel electrochemical biosensing platform, and specifically comprises the following steps:
and 4, outputting the signal of the electrochemical biosensor.
Preferably, the step 1 comprises:
step 1.1, washing the SPCE by using 10mM phosphate buffer solution;
step 1.2, 50uL of chloroauric acid solution with mass concentration of 0.1% is dripped on the SPCE;
step 1.3, setting parameters of a 16-channel electrochemical detector for standby: initial potential: -0.2V, sampling interval: 0.1s, run time: 300s, current sensitivity: 1 x 10-3A/V, rest time: 2 s.
Preferably, step 2 comprises fixing the frame nucleic acid DNA and the single-stranded DNA probe, and the specific operation steps are as follows:
step 2.1, respectively dripping the frame nucleic acid DNA and the single-stranded DNA probe on an SPCE working electrode, and standing overnight in a wet box at room temperature;
step 2.2, washing the SPCE by 10mM phosphate buffer solution;
step 2.3, 50uL of casein with the mass concentration of 1% is dripped on the SPCE, and the mixture is incubated for 2h at 37 ℃.
Further, preferably, the concentration of the framework nucleic acid DNA and the single-stranded DNA probe is 1uM, and the addition volume is 5 uL.
Preferably, the step 3 comprises:
step 3.1 synthesis of Cas12a/crRNA complex;
step 3.2 mix target solution with Cas12a/crRNA complex;
step 3.3, dripping 8 mu L of the mixture obtained in the step 3.2 into an SPCE array modified by FNAs;
step 3.4 SPCE was washed with 10mM PBS buffer and added dropwise to the working electrode with SA-HRP 3uL diluted with 1% casein and incubated at 25 ℃ for 15 minutes.
Further, preferably, the step 3.1 comprises: cas12a was synthesized at a concentration of 60nM, 60nM crRNA, 1 u/. mu.L of RNase inhibitor and 10 XNEB buffer in nuclease-free water to synthesize a Cas12a/crRNA complex.
Preferably, the step 4 comprises: the SPCE was then washed with 10mM PBS, 50. mu.L of TMB solution was dropped onto the electrode, the SPCE was measured at a scanning rate of 100mv/s in the range of-0.3 v to 0.6v, the SPCE was measured at a voltage of 0.05v, and the current was measured at a steady state of 100 s.
The invention also relates to the use of the HPV-16 nucleic acid analysis method for simultaneous detection of viruses, free tumor DNA, miRNA and one or more disease-related biomarkers.
The invention has the advantages that:
FNAs can reduce mass transfer and surface crowding effects, fixing FNAs on electrodes can optimize the recognition between DNA probes extending upward from the FNAs and target molecules, effectively minimize non-specific interactions on the electrochemical interface, and improve sensitivity and stability of detection.
2. By simply altering the DNA probe sequences extending from the FNAs, extended applications to the detection of other viruses, free tumor DNA, miRNA and disease-related biomarkers can be achieved.
3. The current is collected by using the 16-channel electrochemical biosensing platform, the nucleic acid detection does not need a signal amplification process, the ultrahigh sensitivity can be obtained, and simultaneously, the simultaneous detection and analysis of various nucleic acids can be realized.
Drawings
FIG. 1 is a flow chart of the present invention for detecting HPV-16 model targets;
FIG. 2 is a schematic diagram showing the results of the HPV-16 model target detection sensitivity test according to the present invention;
FIG. 3 is a schematic diagram showing the results of the HPV-16 model target detection stability test of the present invention.
Detailed Description
The following detailed description is made with reference to the accompanying drawings.
A CRISPR/Cas12a system established for the first time is utilized to perform cis-trans cutting on a two-phase interface supported by Frame Nucleic Acid (FNAs), so that a novel electrochemical biosensing platform is constructed.
We applied this platform to single-stranded HPV-16 model target detection, achieving a limit of detection (LOD) as low as 100fM under optimized conditions. And a self-made 16-channel electrochemical workstation is adopted, so that the simultaneous analysis of a plurality of samples is realized. Furthermore, our biosensor was shown to be compatible with serum samples, showing great transformation potential in clinical applications, and could be used for early diagnosis of disease.
The principle of the present invention as shown in FIG. 1 is that biotin modified probe DNA extending from tetrahedral FNAs is designed as reporter DNA, which can hybridize with target DNA, biotin modified reporter DNA can be cleaved efficiently by cis and trans cleavage activity of Cas12a, immobilized FNAs can ensure DNA reporter probe with good density and direction control, thereby generating efficient hybridization, making Cas12a more accessible to reporter probe and ensuring efficient cleavage activity thereof.
EXAMPLE 1 the present invention is used in HPV-16 model target detection sensitivity experiment
(1) Synthesis of nano material AuNFs
50uL of chloroauric acid solution with the mass concentration of 0.1% is dripped on the cleaned screen printing electrodes to ensure that each three-electrode system is completely covered.
The parameters of a chronoamperometry method of a 16-channel electrochemical detector are respectively set as follows: -0.2V, sampling interval: 0.1s, run time: 300s, current sensitivity: 1*10-3A/V, rest time: 2 s. And after the operation time is finished, cleaning the electrode for standby.
(2) Fixing tetrahedral FNAs, single-stranded DNA probe:
the synthesized 1uM concentration of frame nucleic acid DNA, single-stranded DNA probe was dropped 5uL onto the working electrode and placed in a wet box overnight at room temperature.
SPCE was washed with 10mM Phosphate Buffered Saline (PBS) (pH 7.4) buffer and incubated with 50uL of 1% casein (w/v) at 37 ℃ for 2 h.
The FNAs modified SPCE can be stored at 4 deg.C for use.
(3) Non-specific cleavage process of CRISPR/Cas12a system
Cas12a/crRNA complex was synthesized by mixing 60nMCAS12a, 60nMCrRNA, 1 u/. mu.LRNA enzyme inhibitor, and 10 XNEB buffer in nuclease-free water at final concentrations.
Different concentrations of target solution (HPV-16) were prepared from the above solutions using a 10-fold gradient dilution method, including PBS solution, 100fM, 1pM,10pM,100pM,1nM,10nM,100 nM.
Immediately, 8. mu.L of Cas12a/crRNA and target mix were dropped onto the FNAs-modified SPCE array and a cleavage event was performed at 37 ℃ for 3 hours.
SPCE was washed with 10mM PBS (pH, 7.4) buffer, and SA-HRP 3uL diluted with 1% casein was added dropwise to the working electrode and incubated at 25 ℃ for 15 minutes.
(4) Signal output of electrochemical biosensor
The SPCE was then rinsed with 10mM PBS (pH 7.4) and 50. mu.L of TMB solution was dropped onto the electrode.
SPCE was measured at a scan rate of 100mv/s, at a voltage of 0.05v, and at a steady state of 100s, in the range-0.3 v to 0.6 v.
As shown in fig. 2, a) CV and B) IT detected different concentrations of HPV-16 target on the interface supported by FNAs by cis and trans cleavage of CRISPR/Cas12a system. From top to bottom: 0.1 pM,100pM and 10 nM. C) FNAs and D) cis-trans cleavage activity of ssDNA probes by CRISPR/Cas12a system,% Δ I is plotted against HPV-16 concentration. E) Delta I% of single-stranded DNA and FNAs electrochemical biosensors in HPV-16DNA series. F) Comparison of the cleavage activity by the CRISPR/Cas12a system FNAs and ssDNA modified interfaces were used to test the effect of HPV-16. The results show that under optimized conditions, the present invention achieves a limit of detection (LOD) for single-stranded HPV-16 as low as 100 fM.
Example 2 the invention is used in HPV-16 model target detection stability experiment
Steps (1) - (4) can be performed with reference to example 1, wherein the target solution is prepared during the non-specific cleavage of the CRISPR/Cas12a system of step (3) as follows:
7 blanks and 10nM target solution (HPV-16) were prepared, once daily on days one to seven, respectively.
The results are shown in FIG. 3: A) the number of mismatches in the target strand includes PAM and adjacent regions. Bold positions represent base mismatches. B) Evaluation of different numbers of mismatches in biosensors supported by FNAs. The concentration of target and mismatched DNA was 1 nM. C) Detection of the FNAs-supported HPV-16 biosensor in different serum concentrations. D) The stability of the FNAs-supported biosensing platform was studied for 0-7 day storage time. The inset represents the blank and current values for 1nM HPV-16 target. The invention realizes the high-stability detection of the single-chain HPV-16.
Claims (8)
1. A method for analyzing nucleic acid, comprising the steps of:
step 1, synthesizing nano material AuNFs;
step 2, constructing a tetrahedral FNAs modified SPCE interface;
step 3, a CRISPR/Cas12a system cutting process;
and 4, outputting the signal of the electrochemical biosensor.
2. A method for analyzing nucleic acid according to claim 1, wherein the step 1 comprises:
step 1.1, washing the SPCE by using 10mM phosphate buffer solution;
step 1.2, 50uL of chloroauric acid solution with mass concentration of 0.1% is dripped on the SPCE;
step 1.3, setting parameters of a 16-channel electrochemical detector for standby: initial potential: -0.2V, sampling interval: 0.1s, run time: 300s, current sensitivity: 1*10-3A/V, rest time: 2 s.
3. A method for analyzing nucleic acids according to claim 1, wherein the tetrahedral FNAs of step 2 comprise a frame nucleic acid DNA and a single-stranded DNA probe, and the specific operation steps are as follows:
step 2.1, dripping the frame nucleic acid DNA and the single-stranded DNA probe on the SPCE working electrode, and standing overnight in a wet box at room temperature;
step 2.2, washing the SPCE by 10mM phosphate buffer solution;
step 2.3, 50uL of casein with the mass concentration of 1% is dripped on the SPCE, and the mixture is incubated for 2h at 37 ℃.
4. An HPV-16 nucleic acid assay method according to claim 3 characterised in that the framework nucleic acid DNA and single stranded DNA probes are present at a concentration of 1uM and in an additive volume of 5 uL.
5. A method for analyzing nucleic acid according to claim 1, wherein said step 3 comprises:
step 3.1 synthesis of Cas12a/crRNA complex;
step 3.2 mix target solution with Cas12a/crRNA complex;
step 3.3, taking 8 mu L of the mixture obtained in the step 3.2, and dripping the mixture into an SPCE array modified by FNAs;
step 3.4 SPCE was washed with 10mM PBS buffer and added dropwise to the working electrode with SA-HRP 3uL diluted with 1% casein and incubated at 25 ℃ for 15 minutes.
6. A method of nucleic acid analysis according to claim 5, characterised in that step 3.1 comprises: cas12a was synthesized at a concentration of 60nM, 60nM crRNA, 1 u/. mu.L of RNase inhibitor and 10 XNEB buffer in nuclease-free water to synthesize a Cas12a/crRNA complex.
7. A method for analyzing nucleic acid according to claim 1, wherein said step 4 comprises: the SPCE was then washed with 10mM PBS, 50. mu.L of TMB solution was dropped onto the electrode, the SPCE was measured at a scanning rate of 100mv/s in the range of-0.3 v to 0.6v, the SPCE was measured at a voltage of 0.05v, and the current was measured at a steady state of 100 s.
8. Use of the nucleic acid analysis method according to any of claims 1 to 7 for the simultaneous detection of viruses, free tumor DNA, miRNA and one or more disease-related biomarkers.
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| CN115901911A (en) * | 2023-01-06 | 2023-04-04 | 南京邮电大学 | Detection method for detecting cardiac troponin I based on CRISPR/Cas12a |
| CN116411135A (en) * | 2023-03-03 | 2023-07-11 | 四川大学 | Inductively Coupled Plasma Mass Spectrometry Nucleic Acid Detection Method Based on DTN and CRISPR Cas12a |
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Cited By (4)
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| CN115228517A (en) * | 2022-03-17 | 2022-10-25 | 烟台大学 | Rotary microfluidic paper chip for virus detection based on framework nucleic acid and preparation method thereof |
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| CN116411135A (en) * | 2023-03-03 | 2023-07-11 | 四川大学 | Inductively Coupled Plasma Mass Spectrometry Nucleic Acid Detection Method Based on DTN and CRISPR Cas12a |
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